Moderately Halophilic Bacteria from Solar Salt Pans of Ribander, Goa: a Comparative Study
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International Journal of Advanced Biotechnology and Research ISSN 0976-2612, Online ISSN 2278–599X , Vol 3, Issue 3, 2012, pp 635-643 http://www.bipublication.com MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA: A COMPARATIVE STUDY 1 2 3 Vaishali V Surve, Meena U Patil , Smita M Dharmadekari 1Department of Biotechnology, Vivekanand College Aurangabad, Maharashtra, India-431001 [email protected] 2Department of Zoology, Dr.BAMU, Aurangabad Maharashtra, India-431001 3Department of Microbiology, Institute of Science, Aurangabad, Maharashtra, India-431001 [Received-08/07/2012, Accepted-01/08/2012] ABSTRACT Hypersaline environment of solar Salt pans are habitats of robust and diverse halophilic microorganisms .The present study makes an effort to isolate and compare the variation and species specific characteristic of the bacteria , isolated from the solar saltpans of Ribander, Goa.The moderate halophiles are studied considering their potential enzymatic applications. Comparative analysis yield valuable information of the extreme ecosystems .Besides, solar saltpans are endangered ecosystems, harbouring potential microbes. This study makes an effort to create awareness for conservation of this unique ecosystem. Solar saltpan water was used as a source for isolation after enrichment of the culture . Morphological , Biochemical, Antibiotic sensitivity, physiological and enzymatic(Protease )characteristics of the isolates were compared. The 16S rRNA sequencing and G+C analysis were used to identify and differentiate halophilic bacterial strains .The 2 moderately halophilic bacteria isolated were phylogenetically similar to Alkalibacillus HSD20 and Virgibacillus panthotheticus, speceis .The isolated bacteria displayed promising potential for protease production and varied species specific characters .The Ribander solar saltpans are a treasure of halophilic bacteria with a sparkling future. The study encourages further exploration of these extremophiles, their haloenzymes and saline habitats . Key words: Solar salterns, Moderately halophilic bacteria, Protease, species specificity [1]INTRODUCTION and salt marshes,or as artificial solar salterns Hypersaline environments are widely [1].Microorganisms that thrive in these distributed on the earth’s continent where they environments have been broadly classified exist either as natural water bodies such as into halophilic microorganism (that is, require permanent saline lakes, ephemeral saltpans salt for their viability) and halotolerant MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA microorganisms which are able to grow in the to NaCl saturation. Characteristic salt-adapted absence as well as in the presence of NaCl. microbial communities are found along the Halophiles can be further divided into slight salinity gradient[6] . Solar saltpans are found halophiles that grow optimally in 3% (w/v) worldwide. Because of the increasing salinity total salt, moderate halophiles optimal growth of the environment, solar salterns are at 3 - 15% (w/v) salt and extreme halophiles considered extreme environments with very that grow optimally at 25% (w/v)salt [ 2 ]. restricted biology [7]. It is surmised that Halophilic and extremely halotolerant marine environments like solarsaltern may microorganisms are present in each of the yield newer strains which may prove to be a three domains of life: archaea, bacteria and rich resource of newer bioactive metabolites eukarya .The domain bacteria typically [8]. However, in spite of a growing interest in contains many types of halophilic and the use of halophilic enzymes for halotolerant microorganisms that spread over a biotechnological applications, there are large number of phylogenetic subgroups. Most relatively few reports in the literature about of these are moderate rather than extreme their production and characterization [9].As halophiles [3]. Scientific interest in industrial process conditions are harsh, there extremophilic microorganisms, especially are demands for biocatalysts that can hyperthermophiles, thermoacidophiles, withstand the process conditions. The majority archaebacterial anaerobes, and of the enzymes used to date originate from hyperhalophiles, has recently increased .One mesophilic organisms and, despite their many reason for this interest is the need to advantages, the application of these enzymes understand the biochemical mechanisms is restricted due to their limited stability at the involved under extreme conditions because of extremes oftemperature, pH and ionic possible biotechnological use of enzymes and strength. On the other hand, extremophiles are molecules from such organisms. Among a potent source of extremozymes,which show extremophilic bacteria, thermophiles are the outmost stability under extreme conditions. most intensively studied. In contrast, less Extremozymes have great economic potential attention has been paid to halophilic in many industrial processes( e.g. agriculture, microorganisms [4]. Halophilic and food, feed and drinks, detergents, halotolerant bacteria secrete a wide range of textile,leather, pulp and paper[10]. Out of the hydrolytic enzymes into their surrounding vast pool of Extremozymes, halophilic environment. Several of these enzymes which proteases are the most widely exploited include amylases, proteases, xylanases and enzymes in the processing of food, leather and cellulases display polyextremophilic detergents [11].Proteases are hydrolytic properties. They are generally haloalkaliphilic enzymes, which can degrade different protein and thermotolerant which renders them sources, so find potential application for waste amenable to an array of industrial processes, treatment, bioremediation, wool quality normally performed at extreme conditions of improvement, meat tenderization, in food, temperature and pH[ 5]. Multi-pond solar leather, pharmaceutical and detergent salterns, which are used worldwide for salt industries[12]. production along tropical and subtropical Goa’s traditional salt industry is said to have coastal areas, present an environment with been a major supplier of salt to the country increasing salt concentrations, from seawater and an exporter to some foreign countries Vaishali V Surve, et al. 636 MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA since the 10 th century.Sparked by the area of this saltpan is 12,329.12m 2 (Figure 1 ) availability of microbial diversity data in these The site is subjected to heavy annual rains(125 salterns and the potentials of moderately cm) during the monsoons. The prevailing halophilic bacteria , an effort is made to study climate is characterized by three distinct and compare the species . Research aims at seasonsviz. Pre-monsoon: from February to making an effort to conserve these unique May; Monsoons: from June to September and microflora for biotechnology research and PostMonsoon: from October to January. The industrial use. climate on an average is generally warm and [II] MATERIALS AND METHODS humid,fluctuating from a minimum of 20 0C in 0 The traditional salt industry has been existing the month of December to 42 C in May. in Goa since 500 A.D. Goan salt was Figure-1 Study area map: Shaded area depicts the considered to be of the best quality and was location of Ribander salterns exported to several African and Arabian countries during the post Medieval period. 2.2Sample Collection -Overlaying saltpan Goa has over 200 salt pans and produces water was collected in sterile glass bottles and o 35,000 metric tones of salt annually[13]. stored at 4 C. Samples were analysed within a 2.1Sampling site : The study site was the 24 hours of collection.The sample collection Ribandar saltpans (15 0 30.166 N and 73 0 was performed as specified in IS:162- 51.245 E) Goa, India and is situated along the 1981[14]. Mandovi estuary in Tiswadi taluka. The total Vaishali V Surve, et al. 637 MODERATELY HALOPHILIC BACTERIA FROM SOLAR SALT PANS OF RIBANDER, GOA 2.3Enrichment and isolation of halophiles - and subsequently measuring their optical Halophiles were enriched in halophilic density[16].Salt concentration was maintained broth(Abrram gibbons media) containing at 15% and pH at 7.0. (gm/lit): Casein acid hydrolysate-5, Yeast 2.6 Antibiotic susceptibility test extract- 10, Proteasepeptone-5, Trisodium Halophilic bacteria are suseptable to citrate- 3, Potassiumchloride- 2, Magnesium antibiotics unlike the halophilic archaea . This sulfate- 20, Fe SO4-0.05 ,Sodiumchloride- character also differentiates the halophilic 150(15%), pH- 7.0 for 10-12 days [15]. From bacteria and archaea.Test for antibiotic enriched 15 % NaCl (w/v) halophilic broth, sensitivity was performed by the Kirby-Bauer cultures were streaked on respective agar disc method[16].The antibiotics tested were media by four sector method for the purpose Penicillin, Gentamycin,Streptomycin and of isolation into pure culture[16]. chloramphenicol.All in a concentration of 2.4Characterization of organisms -Gram 10mcg/disc(Himedia) staining ,Cell morphology and motility were 2.6 Genotypic analysis examined on freshly prepared wet mounts by A]16srRNA sequencing light microscopy. Colony pigmentation was The 16S rRNA genes were isolated from the recorded on agar plates after 10 days of cells PCR amplifications of the 16S rRNA growth was performed [16]. The cultures gene, from the purified genomic DNAs, were were purified on the same salt concentration carried out using the primer sets 27F (5´- and medium from which they were isolated.